Programmable m 6 A modification of cellular RNAs with a Cas13-directed methyltransferase

N 6 -Methyladenosine (m 6 A) is the most widespread internal messenger RNA modification in humans. Despite recent progress in understanding the biological roles of m 6 A, the inability to install m 6 A site specifically in individual transcripts has hampered efforts to elucidate causal relationships between the presence of a specific m 6 A and phenotypic outcomes. In the present study, we demonstrate that nucleus-localized dCas13 fusions with a truncated METTL3 methyltransferase domain and cytoplasm-localized fusions with a modified METTL3:METTL14 methyltransferase complex can direct site-specific m 6 A incorporation in distinct cellular compartments, with the former fusion protein having particularly low off-target activity. Independent cellular assays across multiple sites confirm that this targeted RNA methylation (TRM) system mediates efficient m 6 A installation in endogenous RNA transcripts with high specificity. Finally, we show that TRM can induce m 6 A-mediated changes to transcript abundance and alternative splicing. These findings establish TRM as a tool for targeted epitranscriptome engineering that can reveal the effect of individual m 6 A modifications and dissect their functional roles.